ABSTRACT
Growth differentiation factor 15 (GDF15; also known as MIC-1) is a divergent member of the TGF-ß superfamily and is associated with body-weight regulation in humans and rodents. However, the cognate receptor of GDF15 is unknown. Here we show that GDF15 binds specifically to GDNF family receptor α-like (GFRAL) with high affinity, and that GFRAL requires association with the coreceptor RET to elicit intracellular signaling in response to GDF15 stimulation. We also found that GDF15-mediated reductions in food intake and body weight of mice with obesity were abolished in GFRAL-knockout mice. We further found that GFRAL expression was limited to hindbrain neurons and not present in peripheral tissues, which suggests that GDF15-GFRAL-mediated regulation of food intake is by a central mechanism. Lastly, given that GDF15 did not increase energy expenditure in treated mice with obesity, the anti-obesity actions of the cytokine are likely driven primarily by a reduction in food intake.
Subject(s)
Eating/drug effects , Energy Metabolism/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/drug effects , Growth Differentiation Factor 15/pharmacology , Obesity/metabolism , Weight Loss/drug effects , Animals , Eating/genetics , Energy Metabolism/genetics , Flow Cytometry , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , HEK293 Cells , Humans , In Vitro Techniques , Mice , Mice, Knockout , Rats , Rats, Sprague-Dawley , Surface Plasmon Resonance , Weight Loss/geneticsABSTRACT
Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein. In this format, individual ECD can be produced as a soluble protein or displayed on cell surface, depending on the applied heavy-chain Fab configuration. The unique design of the Fab fusion concept used in the library led to not only superior success rate of protein production, but also versatile applications in various high-throughput screening paradigms including protein-protein binding assays as well as cell binding assays, which were not possible for any other existing expression libraries. The protein library was screened against human coagulation factor VIIa (FVIIa), an approved therapeutic for the treatment of hemophilia, for binding partners by AlphaScreen and ForteBio assays. Two previously known physiological ligands of FVIIa, tissue factor (TF) and endothelial protein C receptor (EPCR) were identified by both assays. The cell surface displayed library was screened against V-domain Ig suppressor of T-cell activation (VISTA), an important immune-checkpoint regulator. Immunoglobulin superfamily member 11 (IgSF11), a potential target for cancer immunotherapy, was identified as a new and previously undescribed binding partner for VISTA. The specificity of the binding was confirmed and validated by both fluorescence-activated cell sorting (FACS) and surface plasmon resonance (SPR) assays in different experimental setups.
Subject(s)
Membrane Proteins , Peptide Library , Receptors, Cell Surface , Recombinant Fusion Proteins , Cloning, Molecular , HEK293 Cells , High-Throughput Screening Assays , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
PURPOSE: Multidrug resistance (MDR) continues to be a major obstacle for successful anticancer therapy. One of the principal factors implicated in MDR is the over expression of P-glycoprotein (Pgp), the product of the MDR1 gene. METHODS: Here we explore the possibility of using the transcription inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) to inhibit Sp1-regulated MDR1 gene expression and restore doxorubicin and paclitaxel sensitivity to multidrug resistant human cancer cells in vitro and in vivo. RESULTS: We found that M4N acted synergistically with doxorubicin and paclitaxel in inhibiting the growth of the cells in culture allowing significant dose reductions of both drugs. We observed no such synergism when M4N was used in combination with cisplatin, another chemotherapeutic agent, but not a Pgp substrate, as analyzed by the combination index and isobologram methods. Analysis of MDR1 mRNA and Pgp levels revealed that at sublethal doses, M4N inhibited MDR1 gene expression in the multidrug resistant NCI/ADR-RES cells and reversed the MDR phenotype as measured by Rhodamine-123 retention. In addition, M4N was found to inhibit doxorubicin-induced MDR1 gene expression in drug sensitive MCF-7 breast cancer cells. CONCLUSIONS: M4N and maltose-tri-O-methyl nordihydroguaiaretic acid (maltose-M3N), a water-soluble derivative of NDGA, were also able to reverse the MDR phenotype of the tumor cells in a xenograft model system and combination therapy with M4N or maltose-M3N and paclitaxel was effective at inhibiting growth of these tumors in nude mice.
Subject(s)
Doxorubicin/pharmacology , Drug Resistance, Multiple/drug effects , Masoprocol/analogs & derivatives , Masoprocol/pharmacology , Monosaccharides/pharmacology , Paclitaxel/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Dose-Response Relationship, Drug , Doxorubicin/administration & dosage , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Gene Expression/drug effects , Humans , Masoprocol/administration & dosage , Mice , Mice, Nude , Monosaccharides/administration & dosage , Paclitaxel/administration & dosage , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Xenograft Model Antitumor Assays/methodsABSTRACT
PURPOSE: We have previously shown that the transcriptional inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) induces growth arrest in tumor cells and exhibits tumoricidal activity when injected intratumorally into tumor cell explants in mice. The experiments reported here were designed to determine whether M(4)N can be given systemically and inhibit the growth of five different human xenograft tumors. EXPERIMENTAL DESIGN: Nude (nu/nu) mice bearing xenografts of each of five human tumor types (i.e., hepatocellular carcinoma, Hep 3B; prostate carcinoma, LNCaP; colorectal carcinoma, HT-29; breast carcinoma, MCF7; and erythroleukemia, K-562) were treated with M4N given i.v. or i.p. in a Cremophor EL-based solvent system or orally in a corn oil based diet. Tumors from the treated animals were measured weekly and analyzed for the expression of the Cdc2 and survivin genes, both previously shown to be down-regulated by M4N. RESULTS: Systemic M4N treatment suppressed the in vivo growth of xenografts in each of the five human tumor types. Four of the five tumor models were particularly sensitive to M4N with tumor growth inhibitions (T/C values) of < or = 42%, whereas the fifth, HT-29, responded to a lesser extent (48.3%). Growth arrest and apoptosis in both the xenograft tumors and in the tumor cells grown in culture were accompanied by reductions in both Cdc2 and tumor-specific survivin gene expression. Pharmacokinetic analysis following oral and i.v. administration to ICR mice indicated an absolute bioavailability for oral M4N of approximately 88%. Minimal drug-related toxicity was observed. CONCLUSION: These preclinical studies establish that when given systemically, M4N can safely and effectively inhibit the growth of human tumors in nude mice.
Subject(s)
Cell Proliferation/drug effects , Masoprocol/analogs & derivatives , Masoprocol/pharmacology , Neoplasms, Experimental/prevention & control , Administration, Oral , Animals , Area Under Curve , CDC2 Protein Kinase/genetics , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HT29 Cells , Humans , Inhibitor of Apoptosis Proteins , Injections, Intravenous , K562 Cells , Masoprocol/administration & dosage , Masoprocol/pharmacokinetics , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Microtubule-Associated Proteins/genetics , Neoplasm Proteins , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survivin , Tissue Distribution , Xenograft Model Antitumor Assays/methodsABSTRACT
BACKGROUND AND PURPOSE: Although hypoxia-inducible factor-1alpha (HIF-1alpha) plays a major role in the prevention of myocardial ischemia, the temporal and spatial patterns of expression of HIF-1alpha in myocardial ischemia-reperfusion are not well known. This study examined the role of HIF-1alpha and vascular endothelial growth factor (VEGF) in myocardial ischemia-reperfusion. METHODS: Adult Wistar rats were studied after ligation of the left anterior descending coronary artery (LAD) for 30 min and then after reperfusion. HIF-1alpha and VEGF were measured immediately after relief of occlusion and at 30 min, 1, 3, 6, and 24 h after reperfusion. HIF-1alpha and VEGF proteins were also measured 6 h after permanent occlusion of the LAD. RESULTS: HIF-1alpha and VEGF mRNA increased 1.8- and 1.4-fold, respectively, immediately after relief of occlusion and reached a maximum of 4.3- and 2.3-fold, respectively, at 3 h after reperfusion and remained elevated up to 24 h. HIF-1alpha and VEGF proteins increased immediately after relief of ischemia. HIF-1alpha protein significantly increased from 0.5 h to 24 h after reperfusion and VEGF protein significantly increased from 1 h to 6 h after reperfusion compared to the sham control. Administration of HIF-1alpha antisense oligonucleotide before ligation of the LAD significantly inhibited VEGF protein expression induced by ischemia-reperfusion. Immunohistochemical study showed increased immunoreactivity of HIF-1alpha and VEGF in the jeopardized myocardium after ischemia-reperfusion. HIF-1alpha and VEGF proteins were increased at 6 h after permanent occlusion of the LAD. CONCLUSIONS: This study demonstrated that HIF-1alpha and VEGF were co-induced in a temporal and spatial pattern after ischemia-reperfusion in the rat ventricular myocardium.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Myocardial Ischemia/metabolism , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion , Vascular Endothelial Growth Factor A/metabolism , Animals , Disease Models, Animal , Rats , Rats, WistarABSTRACT
Blockade of the complement cascade at the C5a/C5a receptor (C5aR)-axis is believed to be an attractive treatment avenue in rheumatoid arthritis (RA). However, the effects of such interventions during the early phases of arthritis remain to be clarified. In this study we use the murine delayed-type hypersensitivity arthritis (DTHA) model to study the very early effects of a blocking, non-depleting anti-C5aR mAb on joint inflammation with treatment synchronised with disease onset, an approach not previously described. The DTHA model is a single-paw inflammatory arthritis model characterised by synchronised and rapid disease onset driven by T-cells, immune complexes and neutrophils. We show that a reduction in paw swelling, bone erosion, cartilage destruction, synovitis and new bone formation is apparent as little as 60 h after administration of a single dose of a blocking, non-depleting anti-mouse C5aR mAb. Importantly, infiltration of neutrophils into the joint and synovium is also reduced following a single dose, demonstrating that C5aR signalling during the early stage of arthritis regulates neutrophil infiltration and activation. Furthermore, the number of T-cells in circulation and in the draining popliteal lymph node is also reduced following a single dose of anti-C5aR, suggesting that modulation of the C5a/C5aR axis results in effects on the T cell compartment in inflammatory arthritis. In summary, these data demonstrate that blockade of C5aR leads to rapid and significant effects on arthritic disease development in a DTHA model strengthening the rationale of C5aR-blockade as a treatment strategy for RA, especially during the early stages of arthritis flare.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Arthritis, Experimental/drug therapy , Edema/drug therapy , Hypersensitivity, Delayed/drug therapy , Receptor, Anaphylatoxin C5a/antagonists & inhibitors , Animals , Antigen-Antibody Complex/metabolism , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Complement C5a/antagonists & inhibitors , Complement C5a/genetics , Complement C5a/immunology , Disease Models, Animal , Edema/immunology , Edema/pathology , Female , Gene Expression , Hindlimb , Humans , Hypersensitivity, Delayed/immunology , Hypersensitivity, Delayed/pathology , Lymphocyte Count , Mice , Mice, Inbred C57BL , Neutrophil Infiltration/drug effects , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/pathology , Receptor, Anaphylatoxin C5a/genetics , Receptor, Anaphylatoxin C5a/immunology , Signal Transduction , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/pathologyABSTRACT
Antrodia camphorata is a fungus native to Taiwan, and it is considered a precious medicinal agent. We analyzed triterpenoids, polysaccharides and 1,3-ß-D-glucan, three major effective components in A. camphorata extracts (ACE). ACE exhibited a selective cytotoxic effect on BxPC-3 human pancreatic cancer cells. ACE markedly inhibited the migration ability of BxPC-3 cells. Treatment of BxPC-3 cells with ACE resulted in the increase of cells in the sub-G1 phase and G2/M phase arrest. Apoptosis was confirmed by validating phosphatidylserine externalization, the observation of characteristic chromatin condensation, and nuclear DNA fragmentation. ACE induced apoptosis in BxPC-3 cells through a mitochondria-dependent pathway by triggering an appropriate balance of bax/bcl-2, cytochrome c release, activation of caspase-9 and -3, and poly(ADP-ribose) polymerase cleavage. ACE shows great therapeutic potential due to its cytotoxic effects against BxPC-3 cells which include inhibiting cell migration and inducing mitochondria-mediated apoptosis.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antrodia/chemistry , Fruiting Bodies, Fungal/chemistry , Glucans/pharmacology , Pancreatic Neoplasms/physiopathology , Polysaccharides/pharmacology , Triterpenes/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cytochromes c/metabolism , DNA Fragmentation/drug effects , Glucans/chemistry , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Pancreatic Neoplasms/drug therapy , Polysaccharides/chemistry , Triterpenes/chemistryABSTRACT
PEGylation modification has been used to improve the pharmacokinetic properties of protein-based drugs. For example, PEGylated human growth hormone (hGH) has been shown to exhibit better pharmacokinetic profiles than the unmodified hGH. Unlike chemical PEGylation of hGH that is difficult to be controlled to result in homogeneity, microbial transglutaminase (mTGase) only conjugates poly(ethelene glycol) (PEG) on glutamine-40 (Q40) and glutamine-141 (Q141) of hGH, the only glutamine residues exposed. Yet, an mTGase that can selectively conjugate PEG to only 1 glutamine residue is more desirable to control the homogeneity of the product. In this study, the authors have developed a novel high-throughput assay, with which they have identified 5 mTGase mutants that are highly specific for conjugating PEG to Q141 of hGH. In this scintillation proximity assay (SPA)-based method, the authors have (1) achieved a high expression level of active mTGase, which is toxic to the living cell, directly from Escherichia coli (0.2 U/mL/OD600) by in vivo activation; (2) developed a high-throughput affinity purification method to eliminate the strong interference of cellular protein to mTGase reaction; and (3) used therapeutic protein as the substrate. This method is highly sensitive, is easily automated, and could be generally applied to screening mTGases with desired specificity targeting on different therapeutic proteins.
Subject(s)
Bacterial Proteins/metabolism , Glycine/chemistry , High-Throughput Screening Assays , Human Growth Hormone/metabolism , Transglutaminases/metabolism , Amino Acid Sequence , Electrophoresis, Capillary , Escherichia coli/genetics , Human Growth Hormone/chemistry , Human Growth Hormone/genetics , Humans , Kinetics , Molecular Sequence Data , Mutation , Plasmids , Sensitivity and Specificity , Streptomyces/enzymology , Substrate Specificity/geneticsABSTRACT
Both cell therapy and angiogenic growth factor gene therapy have been applied to animal studies and clinical trials. Little is known about the direct comparison between cell therapy and angiogenic growth factor gene therapy. The goal of this study was to compare the effects of human bone marrow-derived mesenchymal stem cells (hMSCs) transplantation and injection of angiogenic growth factor genes in a model of acute myocardial infarction in mice. The hMSCs were obtained from adult human bone marrow and expanded in vitro. The purity and characteristics of hMSCs were identified by flow cytometry and immunophenotyping. Immediately after ligation of the left anterior descending coronary artery in male severe combined immunodeficient (SCID) mice, culture-expanded hMSCs or angiogenic growth factor genes were injected intramuscularly at the left anterior free wall. The engrafted hMSCs were positive for cardiac marker, desmin. Infarct size was significantly smaller in the hMSCs-treated group than in the angiopoietin-1 (Ang-1) or vascular endothelial growth factor (VEGF)-treated group at day 28 after infarction. hMSCs transplantation was better in decreasing left ventricular end-diastolic dimension and increasing fractional shortening than Ang1 or VEGF gene therapy. Capillary density was markedly increased after hMSCs transplantation than Ang1 and VEGF gene therapy. In conclusion, intramyocardial transplantation of hMSCs improves cardiac function after acute myocardial infarction through enhancement of angiogenesis and myogenesis in the ischemic myocardium. hMSCs are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction. Transplantation of MSCs may become the future therapy for acute myocardial infarction for myocardial regeneration.
Subject(s)
Angiopoietin-1/genetics , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Myocardial Infarction/therapy , Myocardium/metabolism , Vascular Endothelial Growth Factor A/genetics , Animals , Disease Models, Animal , Humans , Immunophenotyping , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, SCID , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardium/cytology , Neovascularization, Physiologic , Ventricular Function, LeftABSTRACT
We previously reported that Sp1-dependent Cdc2 gene expression is inhibited by tetra-O-methyl nordihydroguaiaretic acid (M(4)N) and that M(4)N is likely responsible for causing growth arrest in M(4)N-treated transformed C3 cells. Here, we show that after M(4)N treatment and cell-cycle arrest, expression of the Sp1-dependent survivin gene, a member of the inhibitor of apoptosis family, is also suppressed, and the mitochondrial apoptotic pathway is activated. To confirm that inhibition of Cdc2 and survivin gene expression is necessary for M(4)N-induced growth arrest and apoptosis, we tested the effect of adding Cdc2 and survivin back to M(4)N-treated cells. Cell division was transiently restored in the presence of M(4)N after transfection of an exogenous Cdc2 gene copy under the control of the Sp1-independent cytomegalovirus promoter. Caspase-3 activation was also reduced by 50% and 75% in transiently and stably survivin-transfected C3 cells, respectively. The results suggest that M(4)N induces growth arrest and apoptosis by suppressing Cdc2 and survivin expression, which constitutes the cellular basis of its antitumoric action.
Subject(s)
Apoptosis/drug effects , CDC2 Protein Kinase/antagonists & inhibitors , Masoprocol/analogs & derivatives , Masoprocol/pharmacology , Microtubule-Associated Proteins/antagonists & inhibitors , Animals , CDC2 Protein Kinase/genetics , Caspase 3 , Caspases/metabolism , Cell Division/drug effects , Cell Line , Cell Nucleus/metabolism , Cyclin B/analysis , Cytoplasm/metabolism , Down-Regulation , Gene Expression Regulation/drug effects , Inhibitor of Apoptosis Proteins , Mice , Microtubule-Associated Proteins/genetics , Neoplasm Proteins , SurvivinABSTRACT
The angiopoietins and Tie receptors are involved in blood vessel formation. The role of the angiopoietin/Tie receptor system in myocardial ischaemia/reperfusion is not well known. To investigate the participation of angiopoietins and Tie receptors in myocardial ischaemia/reperfusion, adult Wistar rats were studied in which the left coronary artery was ligated for 30 min, followed by reperfusion. Angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), Tie1 and Tie2 were measured immediately after relief of occlusion, and 1, 6, 24 and 72 h after reperfusion, by Northern blot, Western blot and immunohistochemical staining. Ang2 mRNA was increased significantly at 24 h and 48 h after reperfusion, and returned to baseline levels at 72 h, in the jeopardized myocardium. Tie2 mRNA increased 3.4-fold immediately after the relief of occlusion, reached a maximum 8-fold increase at 24 h after reperfusion and remained elevated up to 72 h. Ang2 protein levels also increased after reperfusion, reaching a maximum 2.2-fold increase at 48 h after reperfusion. Tie2 protein increased immediately after relief of ischaemia, and showed a significant increase from 6 h to 72 h after reperfusion as compared with the sham control. Ang1 and Tie1 mRNA and protein did not show significant changes after ischaemia/reperfusion. Immunohistochemical studies also showed increased immunoreactivity of Ang2 and Tie2 in the jeopardized myocardium after ischaemia/reperfusion. In conclusion, expression of both Ang2 and Tie2 increased after ischaemia/reperfusion in the rat ventricular myocardium, while the expression of Ang1 and Tie1 did not.
Subject(s)
Angiogenesis Inducing Agents/genetics , Myocardial Reperfusion Injury/metabolism , Myocardium/metabolism , Neoplasm Proteins/genetics , Proto-Oncogene Proteins , RNA, Messenger/analysis , Angiogenesis Inducing Agents/analysis , Angiopoietin-1 , Angiopoietin-2 , Animals , Blotting, Northern/methods , Blotting, Western/methods , Immunohistochemistry/methods , Membrane Glycoproteins/analysis , Myocardium/chemistry , Neoplasm Proteins/analysis , Rats , Rats, Wistar , Receptor Protein-Tyrosine Kinases/analysis , Receptor, TIE-1 , Receptor, TIE-2 , Receptors, Cell Surface/analysis , Receptors, TIE , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
One of the biological effects of hyperbaric oxygen (HBO) therapy in enhancing ischemia-related wound healing is the induction of angiogenesis. To elucidate the mechanism(s) underlying the HBO-induced angiogenesis, we studied the expression of several angiogenesis-related genes in human umbilical vein endothelial cells exposed to HBO. Western blot analyses showed that HBO enhanced the expression of angiopoietin-2 (Ang2) with no effect on the expression of Tie2, angiopoietin-1, and VEGF. The induction of Ang2 was further confirmed by immunohistochemistry, quantitative PCR, and Northern blot analyses. Inhibition of endothelial nitric oxide synthase blocked the HBO-induced Ang2 expression, but failed to block hypoxia-induced Ang2 expression. These data indicated that HBO-induced Ang2 expression may be through transcriptional stimulation, and requires the nitric oxide signaling pathway, which may play an important role in HBO-induced angiogenesis.